Preventive and curative effect of edaravone on nerve functions and oxidative stress in experimental diabetic neuropathy.

Oxidative stress is implicated as a final common pathway in the development of diabetic neuropathy and pharmacological interventions targeted at inhibiting free radical production have shown beneficial effects. In the present study, we have investigated the effects of edaravone (3 mg/kg; 3-Methyl-1-phenyl-2-pyrazolin-5-one), a free radical scavenger (relatively selective to hydroxyl radicals) in streptozotocin (50 mg/kg i.p.) induced diabetic neuropathy in male Sprague-Dawley rats. Significant reduction (18%) in motor nerve conduction velocity, nerve blood flow (55%) and tail flick latency in cold (53%) and hot (50%) immersion test was observed in diabetic rats compared to age matched non-diabetic rats. Preventive (8 week) and curative (2 week) treatment of edaravone significantly improved the nerve conduction velocity and nociception but not nerve blood flow in diabetic rats. The changes in lipid peroxidation status and anti-oxidant enzymes (Superoxide dismutase and Catalase) levels observed in diabetic rats were significantly restored by edaravone treatment. Increase in blood pressure and vascular resistance was also significantly attenuated by edaravone treatment. This study provides experimental evidence to preventive and curative effect of edaravone on nerve function and oxidative stress in animal model of diabetic neuropathy. Hence edaravone may be tried clinically for the treatment of diabetic neuropathy since it is clinically used in stroke patients.

Structural analysis of isoform-specific inhibitors targeting the tetrahydrobiopterin binding site of human nitric oxide synthases.

Nitric oxide synthesized from l-arginine by nitric oxide synthase isoforms (NOS-I-III) is physiologically important but also can be deleterious when overproduced. Selective NOS inhibitors are of clinical interest, given their differing pathophysiological roles. Here we describe our approach to target the unique NOS (6R,1'R,2'S)-5,6,7,8-tetrahydrobiopterin (H(4)Bip) binding site. By a combination of ligand- and structure-based design, the structure-activity relationship (SAR) for a focused set of 41 pteridine analogues on four scaffolds was developed, revealing selective NOS-I inhibitors. The X-ray crystal structure of rat NOS-I dimeric-oxygenase domain with H(4)Bip and l-arginine was determined and used for human isoform homology modeling. All available NOS structural information was subjected to comparative analysis of favorable protein-ligand interactions using the GRID/concensus principal component analysis (CPCA) approach to identify the isoform-specific interaction site. Our interpretation, based on protein structures, is in good agreement with the ligand SAR and thus permits the rational design of next-generation inhibitors targeting the H(4)Bip binding site with enhanced isoform selectivity for therapeutics in pathology with NO overproduction.

Reduced cGMP signaling associated with neointimal proliferation and vascular dysfunction in late-stage atherosclerosis.

Atherosclerosis is associated with alterations in nitric oxide (NO)/cGMP signaling. In early stages of the disease, inflammatory and possibly other cells produce reactive oxygen species that scavenge vasoprotective NO. In addition to the oxidative stress, expression and activity of enzymes downstream to NO formation may also be affected. Here, we show in the aortas of chronically hypercholesterolemic rabbits (a model of late-stage atherosclerosis), both subunits and specific activity of the NO receptor soluble guanylyl cyclase (sGC) were significantly reduced, whereas overall NO synthase activity was unaffected. These changes were most prominent in the neointimal layer, wherein cGMP-dependent protein kinase I (cGK) levels also were reduced. Additionally, a protein (p38(nt)) that was constitutively tyrosine-nitrated was detected, and its expression was significantly reduced in atherosclerotic aorta. Phosphorylation of the cGK substrate vasodilator-stimulated phosphoprotein (VASP) at Ser-239, an established biochemical endpoint of NO/cGMP signaling, also was reduced. Thus, late-stage atherosclerosis is associated not only with enhanced NO breakdown but also with altered NO reception and cGMP signaling. Preferential down-regulation in neointima suggests a direct connection of these changes to neointimal proliferation and vascular dysfunction and provides a rationale for future pharmacotherapy using classical and novel sGC activators.